repeatedly selfing the backcrossed progeny for at least five generations without additional backcrossing to produce a backcrossed Fn progeny (BC1Fn), wherein n>5 and the genetic variability within the Fn generation is 3.125% or less; and

2. The method of claim 1, wherein the backcrossed F1 (BC1F1) progeny for selfing further possess at least one desired trait selected from the group consisting of plant type, fertility, plant height, head type, kernel type, and maturity.

repeatedly selfing the backcrossed progeny for at least five generations without additional backcrossing to produce a backcrossed Fn progeny (BC1Fn) wherein n>5 and the genetic variability within the Fn generation is 3.125% or less; and

11. The method of claim 10, wherein the Fusarium resistant tetraploid (AABB) durum wheat exhibits a mean disease severity in a Type II infection field test assay of less than 32%.

12. The method of claim 10, wherein the Fursarium resistant tetraploid (AABB) durum wheat exhibits a mean disease severity in a Type II infection greenhouse assay of less than 16%.

Description

This application claims the benefit of U.S. Provisional Application Ser. No. 60/577,854, filed 8 Jun. 2004, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under a grant from the United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Grant No. 59-0790-9-033. The U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Durum wheat (the tetraploid wheat Triticum turgidum L. var. durum, synonym T. durum) is one of the most important cereal crops in the world. Also known as “hard” wheat or macaroni wheat, it is cultivated in semiarid regions of the world such as North Africa, Mediterranean Europe, the North American Great Plains and the Middle East. Its kernel size, hardness and golden amber color make it most suitable for manufacturing a unique and diverse range of food products. Pasta and couscous are the most common paste products made from durum wheat.

Durum wheat also can be used for making bread, however, bread wheat (the hexaploid wheat T. aestivum) is the main source of flour for making bread. Generally bread wheat is not used to manufacture pasta or couscous.

Wheat belongs to the genus Triticum, all members of which contain a multiple of the basic haploid set of seven chromosomes (x=7). The wheats form an all polyploid series with diploid (2n=2x=14), tetraploid (2n=4x=28), and hexaploid (2n=6x=42) species. Within each species, chromosomes pair in a diploid-like fashion, and the mode of inheritance is disomic.

Cytogenetic, biochemical, morphological and genetic analyses have been used to assess the evolutionary development of the cultivated tetraploid and hexaploid wheat. The designated A-genome is derived from the diploid T. monococcum L. (synonyms T. boeoticum and T. urartu). T. monococcum was long considered the A-genome donor. At the wild tetraploid level, T. dicocoides (AABB) may have the A-genome from T. monococcum and the B-genome of T. speltoides (Tausch) Gren. Ex Richter, synonym Aegilops speltoides. Common bread wheat, the hexaploid T. aestivum (AABBDD) has the A- and B-genomes of a tetraploid T. turgidum and the D-genome derived from T. taushii (Coss.) Schmal., synonym Aegilops sqarrosa. The two species T. turgidum L. var. dicoccoides and Aegilops sqaurrosa are considered the nearest wild progenitors of common bread wheat. T. dicoccoides is the only wild member of the wheat group fully interfertile with cultivated T. turgidum L var. durum.

The tetraploid emmer wheats T. dicoccum shrank, T. dicoccoides, and T. turgidum L var. durum also can be crossed directly with hexaploid wheats. The F1 generation may exhibit a high degree of sterility, but seed set can be obtained.

Fusarium head blight (FHB) is caused by the fungus Fusarium, typically F. graminearum Schwabe (telomorph Gibberella zea (Schwein.) Petch) but other causal agents can include F. culmorum and F. avenaceum. Fusarium head blight is a serious threat to durum wheat. Since 1993, it is estimated that Fusarium head blight has cost over $3 billion in direct and indirect losses in North Dakota (Sayler, Scab on rampage: where do we go from here? Prairie Grains, November/December, issue 10 pp 14, 19-21, 35 and 39 (1997)). Fusarium head blight not only reduces yield but also reduces the quality of the end products of durum wheat (Dexter et al., Cereal Chem., 74:519 (1997)). The fungus is also associated with mycotoxins, particularly trichothecene deoxynivalenol (DON vomitoxin), that are hazardous to humans and other animals.

There is a continuous decline in harvested durum acreage and production in North Dakota because of Fusarium head blight. The harvested acreage in North Dakota in 2001 was 2.25 million acres. This acreage is 22% less than the year 2000 (State of North Dakota, Agriculture Statistics). In 2001 North Dakota produced 60.75 million bushels of durum wheat, which was a 22% decrease in production as compared to production in the year 2000 (National Agriculture Statistics, 2001). The decline in harvested acreage and durum production in North Dakota is disastrous to the farm economy and has direct impact on the national pasta industry. In addition, the international export market is also greatly affected since North Dakota on average produces 75% of the durum in the United States.

Fungicides can be used to improve yield and other agronomic traits but the level of improvement is below the margin of the economic return (McMullen, Evaluation of fungicides for suppression of Fusarium head blight. in Current research on Fusarium head blight of small grains, November (1997) NDSU research publication, Fargo, N.Dak. (1997)). Although fungicides may reduce Fusarium head blight, the use of genetic resistance is the most environmentally safe and economical way to control the disease. Durum wheat with appropriate combinations of resistant genes could effectively control the disease. Accordingly, what is needed is the development of wheat, particularly durum wheat, that is genetically resistant to Fusarium.

SUMMARY OF THE INVENTION

The invention provides Fusarium resistant tetraploid wheat as well as methods for making and using such wheat. A preferred embodiment of the method of producing Fusarium resistant tetraploid wheat includes crossing Fusarium resistant hexaploid wheat with a tetraploid wheat to produce F1 progeny, backcrossing the F1 progeny with a tetraploid wheat to produce backcrossed F1 (BC1F1) progeny, and selfing the backcrossed F1 (BC1F1) progeny to produce backcrossed progeny (BC1F2) that include the Fusarium resistant tetraploid wheat.

Seeds and other plant parts of Fusarium resistant tetraploid wheat, such as a leaf, stem, root, embryo, meristematic tissue, callus tissue, germplasm, gametophyte, saprophyte, pollen or microspore, are also provided by the invention. Progeny of Fusarium resistant tetraploid wheat plants, including progeny of crosses and backcrosses utilizing Fusarium resistant tetraploid wheat, are also included in the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gray scale for determining Fusarium head blight (FHB) disease severity (% disease severity is indicated on the x-axis) (Stack and McMullen, A visual scale to estimate severity of Fusarium head blight in wheat. No. Dak. St. Univ. Bull. P-1095 (1995)).

FIG. 2 is a gray scale version of color photographs showing FHB severity (% disease severity is indicated on the x-axis) (Stack and McMullen, A visual scale to estimate severity of Fusarium head blight in wheat. No. Dak. St. Univ. Bull. P-1095 (1995)).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is based on the discovery that resistance to Fusarium head blight (FHB) (also referred to herein as “Fusarium resistance” or “FHB resistance”) can be transferred from known Fusarium resistant hexaploid wheat to tetraploid wheat through use of the method of the invention. Accordingly, the invention also provides Fusarium resistant tetraploid wheat, and products thereof, that will help to provide good quality wheat for the future. The Fusarium resistant wheat produced according to the method is produced through use of basic plant breeding and is therefore not a genetically modified organism.

The term “wheat” as used herein includes generally any plant of the wheat genus (Triticum) including wheat species, varieties, subvarieties, hybrids, cultivars, lines, strains and the like.

The Fusarium resistant hexaploid wheat, Sumai 3, is probably the most widely used wheat from which Fusarium resistance is obtained in the world. It has been used in Chinese breeding programs for at least 20 years (Liu, Recent advances in research on wheat scab in China. p. 174-181, in Wheat for more tropical environments. CIMMYT (Centro Internacional de Mejoramiento de Maiz y Trigo), Mexico, D.F. Mexico (1984)) and since introduction into the USA, it has been used by winter and spring wheat breeders (Wilcoxson, Historical review of scab research, p. 1-5, in Proc. (1st) Regional Scab Forum, Moorhead, Minn. Publ. Minn. Wheat Res. & Prom Council, Red Lake Falls, Minn. (1993)). The FHB resistance in Sumai 3 is heritable, stable and consistent across environments.

The Fusarium resistant hexaploid spring wheat, Wnagshuibai, has also been used by wheat breeders in the USA, but has not been as widely used as Sumai 3. The FHB resistance in Wnagshuibai is also heritable, stable and consistent across environments.

While Sumai 3 has been successfully used by hexaploid wheat breeders as a source of Fusarium resistance, tetraploid durum wheat breeders have had no success in using it. This lack of success initially led breeders to believe that the resistance genes from Sumai 3 might be on the D-genome of hexaploid wheat and would therefore not recombine with tetraploid durum wheat where the D-genome is absent (the durum genome is AABB). However, the resistance genes from Sumai 3 have been mapped on the A-genome and the B-genome (Kolb et al., Crop Sci., 41:611 (2001)). It is now believed that the genetic background of the elite durum germplasm may be suppressing the Sumai 3 resistance. Accordingly, it was surprising when resistance from hexaploid Sumai 3 wheat and hexaploid Wangshuibai wheat was successfully transferred to tetraploid elite North Dakota durum wheat germplasm using the method of the invention.

Fusarium Resistance

Fusarium or FHB “resistance” refers to the ability of wheat to resist infection by Fusarium. There are a number of ways to measure resistance to FHB, which include measuring resistance to initial infection, resistance to spreading (Type II resistance), resistance to kernel infection, tolerance and toxin accumulation/degradation. Preferably, FHB resistance is evaluated using an assay for “Type II” resistance, where an infection is introduced into the middle of a stalk and the plant is examined for spreading up or down the stalk. In a resistant plant, the infection will remain localized, perhaps infecting only one floweret or spikelet. Most breeding programs target “Type II” resistance, i.e., resistance to spread of infection within the spike of a plant.

FHB resistance can be evaluated, for example, by examining the plant for disease after exposure to a Fusarium inoculum. Wheat can be planted and inoculated with Fusarium according to methods described herein and known in the art (e.g., Stack, Can. J. Plant Path., 11:137 (1989)). The wheat is then inspected to determine the level (e.g., spreading) of Fusarium infection (disease severity) produced on the wheat. A visual scale, as shown in FIG. 1 and FIG. 2, and as described in the art (e.g., Stack and McMullen, A visual scale to estimate severity of Fusarium head blight in wheat. No. Dak. St. Univ. Bull. P-1095 (1995)) and in Example I below, is commonly used to assess resistance to Fusarium. Fusarium resistance can be tested under a variety of conditions, such as in a nursery setting and/or in a field setting.

Fusarium resistance and disease severity are, of course, inversely related. A high “% disease severity” value indicates that a wheat exhibits low resistance to Fusarium while a low “% disease severity” value indicates that a wheat exhibits high resistance to Fusarium infection.

Preferably the level of disease severity of the Fusarium resistant tetraploid wheat of the invention is less than 32%, more preferably the level of disease severity of the Fusarium resistant tetraploid wheat is less than 20%, most preferably the level of disease severity of the Fusarium resistant tetraploid wheat is less than 10%. Preferably the level of disease severity of the Fusarium resistant tetraploid wheat is between 7% and 30%. More preferably the level of disease severity of the Fusarium resistant tetraploid wheat is between 7% and 20%. Most preferably the level of disease severity of the Fusarium resistant tetraploid wheat is between 7% and 10%.

Preferably a progeny tetraploid wheat to which Fusarium resistance is transferred through crossing with a Fusarium resistant hexaploid wheat exhibits greater Fusarium resistance than the parent tetraploid wheat.

The presence or level of Fusarium resistance can also be assessed through use of molecular markers that are linked to Fusarium resistance. For example, the microsatellite locus Xgwm2 is tightly linked to Fusarium resistance (Otto et al., Plant Molecular Biology, 48:625 (2002)). Other examples of molecular markers that are associated with Fusarium resistance include a major quantitative trait loci that is designated Qfhs.ndsu-3BS, and a simple sequence repeat marker that is designated Xgwm533. Accordingly, wheat can be additionally assessed for Fusarium resistance based on the presence of a molecular marker within the screened wheat. Additional markers that are associated with Fusarium resistance can also be used to screen wheat for Fusarium resistance.

Some durum wheat already exhibit an increased level of Fusarium resistance. For example, the newly released cultivars: Lebsock (Elias et al., Crop Sci., 41:2007 (2001)), Plaza (Elias et al., Crop Sci., 41:2008 (2001)), Maier (Elias and Miller, Crop Sci., 40:1498 (2000)), Belzer (Elias et al., Crop Sci., 39:881 (1999)) and Ben (Elias and Miller, Crop Sci., 38:895 (1998)) have less disease severity and deoxynivalenol (DON) levels than the older cultivars, Renville (Cantrell et al., Crop Sci., 29:1329 (1989)) and Monroe (Cantrell et al., Crop Sci., 26:200 (1986)). However, the level of resistance in these cultivars is still much lower than that found in hexaploid wheat germplasm. Observed disease severities were within a range of 30% to 60%. It has been reported that the durum Langdon dicoccoides 3A substitution line [LDN(DIC-3A)] was less susceptible to FHB, 12.5% to 29.9%, than all the other substitution lines (Stack et al., Crop Sci., 42:637 (2002)). In comparison with the resistance of Sumai 3, LDN(DIC-3A) is characterized as moderately resistant, with a mean disease severity of 19.8%. A microsatellite locus, Xgwm2, is tightly linked to this resistance (Otto et al., Plant Molecular Biology, 48:625 (2002)) and is being used in the durum breeding program at North Dakota State University (NDSU).

Plant Breeding

The method for producing Fusarium resistant tetraploid wheat includes crossing hexaploid Fusarium resistant wheat with tetraploid wheat to produce F1 progeny, backcrossing the F1 progeny with tetraploid wheat to produce a backcrossed F1 (BC1F1) progeny, and selfing the backcrossed F1 progeny to produce backcrossed progeny (BC1F2) that include members that are resistant to Fusarium.

A single cross is a cross between two parents, for example between Sumai 3 (hexaploid) and Sceptre (tetraploid) which is labeled as Sumai 3/Sceptre. Making crosses requires emasculating the female flower and later pollinating it with pollen from the male flower. Forceps are used for emasculation to remove the anthers from the female flower (female parent). The emasculated female flower is covered with glassine bags to avoid out-crossing. Four to five days later when the flower is mature enough to be receptive, pollen is transferred from the male flower. Thirty days later hybrid seed is harvested and planted to produce F1 progenies.

A “backcross,” as that term is used herein, is a cross between (a) an F1 progeny and (b) one of its parents or a variety with one or more similar features of a parent (the latter being sometimes known as a “top cross”). In the present method, the purpose of the backcross is to reconstitute the tetraploid background. For example, after a hexaploid wheat (e.g., Sumai 3) is crossed with a tetraploid wheat (e.g., Sceptre), F1 progeny can be crossed to another tetraploid wheat as a backcross. Sumai 3 can, for example, be crossed to Sceptre, and the resulting F1 (Sumai 3/Sceptre) can be crossed to a tetraploid line D88816. This backcross is labeled as Sumai 3/Sceptre//D88816, with D88816 being used to reconstitute the tetraploid background.

Advantageously, Fusarium resistant tetraploid wheat (for example, the experimental durum lines described in Example I) can be crossed with the germplasm of any tetraploid wheat of interest to produce an FHB resistant plant with additional desired traits. These traits exhibited by the plant can be observable in the plant's phenotype and/or genotype. The progeny of such crosses may exhibit traits such as improved yield, pasta quality and/or robustness. By crossing Fursarium resistant tetraploid lines with other, agronomically acceptable lines, germplasm can be developed that is both agronomically acceptable and disease resistant. The present invention thus encompasses the use of Fusarium resistant tetraploid wheat as a parent in crosses with other tetraploid wheat, as well as the Fusarium resistant progeny of such crosses.

Optionally, members of the backcrossed progeny (BC1F2) are then selected using selection criteria that can include, but are not limited to, plant features such as plant type, fertility, plant height, head type, maturity, kernel type, and the like. Selected members of the backcrossed progeny (BC1F2) are then selfed to produce additional (BC1F3) progeny, which are selected using selection criteria and selfed to produce (BC1F4) progeny. This process is continued until tetraploid wheat is produced that has increased Fusarium resistance, and other plant features that were selected. This process may be repeated until tetraploid wheat is produced having the selected plant features. In some examples, selfed progeny are produced by performing one to seven selfings, one to ten selfings, one to twenty selfings, and single integer selfings thereof. Examples of such single integer selfings include (BC1F6), (BC1F7), (BC1F8), (BC1F9), (BC1F10), (BC1F11), progeny and so on.

In a preferred embodiment of the method of producing Fusarium resistant tetraploid wheat, the F2 population is large in number (e.g., more than 2000 members, preferably more than 3,000 members, most preferably more than 4,000 members) and a relatively large number of those members (e.g., over 100, preferably over 200) are selected for selfing. Families F3, F4, F5, F6, F7, F8, F9, and so on, are also large compared to standard breeding protocols. The large size of the families increases the probability of a recovery of a line that has Fusarium resistance. Selection within a family, even if the family is 98% genetically identical, surprisingly yields plants with genetic differences. Some of these selected plants exhibited Fusarium resistance. Most breeders do not select within F5 families because of the little genetic diversity present in these families, as they have reached 96.875% homozygosity. However, as described herein in Example I, the little genetic variability present in the BC1F5 generation was productively explored.

As shown in Example I, tetraploid wheat produced through transfer of Fusarium resistance from hexaploid wheat to the tetraploid wheat according to this method exhibit a Fusarium disease severity of about 7% to about 16% in greenhouse testing and about 12% to about 32% in field testing.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLESExample IPreparation of FHB Resistant Tetraploid Wheat

Summary

Tetraploid wheat that was not resistant to Fusarium was crossed with Fusarium resistant hexaploid wheat. The progeny were then backcrossed with tetraploid wheat to produce backcrossed F1 (BC1F1) progeny. The backcrossed F1 progeny were then selfed to produce backcrossed progeny (BC1F2) that include the Fusarium resistant tetraploid wheat.

Materials

Sceptre and Medora durum wheat were developed by the Department of Plant Science and Plant Pathology at the University of Saskatchewan and were released on 5 Jul. 1985 and May, 1982, respectively. Sceptre and Medora exhibit high yield and quality but do not exhibit Fusarium resistance. A complete description of Sceptre has been published (Knott, Can. J. Plant Sci., 66:407 (1986)). A complete description of Medora has also been published (Leisle, Can. J. Plant Sci., 66:999 (1986)).

Ben durum wheat was developed by the North Dakota Agricultural Experiment Station in cooperation with USDA-ARS and released in March of 1996. Ben was registered and was protected under the U.S. Plant Variety Protection Act for Foundation, Registered, and Certified seed classes (PVP Certificate no. 9700089) (Elias et al., Crop Science, 38:895 (1998)). Ben durum wheat also does not exhibit Fusarium resistance.

The durum experimental lines D88096, D88816, D88090 and D88690 were developed by the Durum Wheat Breeding Program at North Dakota State University for possible release as varieties. These wheat varieties also do not exhibit Fusarium resistance.

The F1 resulting from the Sumai 3/Sceptre cross was backcrossed to the four durum experimental lines, D88096, D88816, D88090 and D88690, to generate four different backcrosses F1 (BC1F1) Sumai 3/Sceptre//D88096, Sumai 3/Sceptre//D88816, Sumai 3/Sceptre//D88090, and Sumai 3/Sceptre//D88690.

The F1 resulting from the Sumai 3/Medora cross was backcrossed to Medora to generate backcross F1 (BC1F1) Medora//Sumai 3/Medora.

The F1 resulting from the Wnagshuibai/Ben cross was backcrossed to Ben to generate backcross F1 (BC1F1) Wnagshuibai/Ben//Ben.

The purpose of the backcross is to reconstitute the tetraploid background. All BC1F1 wheat were selfed to produce BC1F2 progenies.

The BC1F1 progenies were planted in a field and single head (spike) selections were made from the BC1F2 generation for durum plant type, fertility, plant height, head type, maturity, kernel type, and other agronomic traits. BC1F3 head rows were made following these selections. At the BC1F3, further selections were made from the BC1F3 generation for the same traits described earlier. First selection was practiced among head rows then within each selected head row the best two plants were selected and planted as sister head rows in the next generation. The BC1F4 head rows were made from these selections. A similar selection procedure to the BC1F3 generation was practiced to develop the BC1F5 generation.

The BC1F5 generations were planted as head hill plots (20 seed/hill) in a field Fusarium head blight nursery at Prosper, N.Dak. for FHB evaluation and selection.

Corn colonized with F. graminearum (grain spawn) was used as a source of inoculum in the nursery. The grain spawn was spread onto the ground by hand at a rate of 40 grams/meter2. The first spawn was spread when the durum wheat was about two weeks from flowering. Additional fresh spawn was spread when needed. The nursery was equipped with a misting system to keep humidity at optimum level for disease development.

First selection was practiced for FHB resistance among head hill plots. Then, within each selected hill plot, the best six plants were selected and planted as sister head rows (BC1F6) in the next generation.

The BC1F6 generations were planted as head rows in a Fusarium head blight nursery at the Academy of Agricultural Sciences, Plant Protection Institute Shanghai, China, (AASPPIS). Twenty kernels from each accession were planted in single 1.5 meter long rows. Entries were assigned to experimental units using a modified augmented block design. Two to three weeks prior to flowering, rice and wheat kernels infected with F. graminearum were spread by hand onto the ground to create an artificial epidemic. The nursery was equipped with a misting system to keep humidity at the optimum level for disease development. Resistant rows were selected and then BC1F7 heads from the selected head rows were selected, threshed, and shipped back to North Dakota State University.

The BC1F7 heads were planted as head rows in the nursery at North Dakota State University for FHB evaluations. Methods of inoculum preparation and inoculation in the nursery that were used are known (Stack, Can. J. Plant Path., 11:137 (1989)). The single spikelet injection method was used in which the inoculum is injected into a single spikelet near the middle of the spike near anthesis. Plants were misted periodically to maintain high humidity for disease development. Plants were rated for Type II disease severity 3 to 3.5 weeks after inoculation using a known scale as described herein (Stack and McMullen, A visual scale to estimate severity of FHB in wheat. No. Dak. St. Univ. Bull. P-1095 (1995)). Mean Type II disease severity of progenies from these crosses are presented in Table 2. Selected lines that are in Table 2 were planted as a randomized complete design trial with four replicates in the field in 2002 for evaluations relying on natural epidemic.

The trial was also planted in the field Fusarium head blight screening nursery at Prosper, N.Dak. Environmental conditions in 2002 were favorable for inducing a severe natural FHB epidemic. The natural epidemic provided good data for the trial that was not in the screening nursery. Data from this trial is presented in Table 3. Fusarium head blight Type II disease severity ratings of these lines in the trial ranged from 13% to 31.5%.

All lines generated from the crosses were checked for chromosome number to insure their ploidy level and check for any abnormalities such as monosomics or chromosome additions. Seeds of the durum wheat lines were germinated on wet filter paper in a petri-dish at 25° C. Roots that were 2-3 cm long were collected and treated in ice water for 20 hours. The roots were then fixed in a solution of 3:1 (95% alcohol:glacial acetic acid). The roots were stained with 2% acetocarmine at room temperature for 1-2 hours before chromosome preparation. Mitotic chromosomes were prepared following known procedures (Cai and Liu, Theor. Appl. Genet., 77:81 (1989)). Mitotic chromosomes in each of the durum lines were counted under an Olympus microscope. All lines were found to have 14 pairs of chromosomes without any abnormalities indicating that they are tetraploid wheat.

Molecular Markers for FHB Resistance

Identification of DNA markers associated with FHB resistance is thought to be a useful tool for wheat breeders working on developing FHB resistant wheat germplasm. A considerable number of mapping studies have been conducted on the Type II resistance of Sumai 3 and its derivatives. A major quantitative trait loci (QTL) was identified in Sumai 3 and designated as Qjhs.ndsu-3BS that is widely used by wheat breeders in the United States. A SSR (Simple Sequence Repeats) marker Xgwm533 that explains 41.6% of the variation of FHB resistance associated with this QTL has been identified (Anderson et al., Theor. Apl. Genet., 102:1161 (2001)). Many breeding programs are using the Xgwm533 marker to check the presence of this QTL in their germplasm. The Xgwm533 was also used to check the presence of the Sumai 3 QTL in the progenies of the hexaploid by tetraploid crosses.

For DNA extraction, a Flinders Technology Associates (FTA) plant purification protocol was used. Leaf tissue was collected at the three leaf stage and smashed onto the FTA cards. Cell membranes and organelles in the leaf tissue were lysed and DNA becomes entrapped in the fibers of the FTA matrix due to being smashed onto the FTA cards. A 2.0 mm punch from within the middle of the smashed leaf stain was removed using a 2.0 mm Harris Micro Punch tool and transferred to an appropriate PCR amplification tube. Each punch was washed twice with 200 μL of FTA reagent followed by an equal number of washings with TE 10 mM Tris-Hcl pH 8.0; 0.1 mM EDTA ph 8.0. The punch was then dried at room temperature for 3 hours and then used for PCR amplification. The presence or absence of the marker in the lines is reported in Table 2 and Table 3.

The complete disclosures of all patents, patent applications including provisional patent applications, and publications, and electronically available material (e.g., GenBank amino acid and nucleotide sequence submissions) cited herein are incorporated by reference. The foregoing detailed description and examples have been provided for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described; many variations will be apparent to one skilled in the art and are intended to be included within the invention defined by the claims.